The present invention relates to processes for continuously obtaining extracellular or secreted enzymes (exoenzymes) by cultivation of exoenzyme-producing bacteria in a continuous-flow fermenter.
Exoenzymes such as proteases, xylanases and amylases are obtained by cultivation of bacteria, in particular of strains of the genus Bacillus. To date, exoenzymes have been prepared only by discontinuous (batch) fermentation, because the secretion of these enzymes by the microorganisms rapidly diminishes in continuous culture (chemostat). See, generally, Priest, "Extracellular Enzyme Synthesis in the Genus Bacillus," Bacteriol. Rev. 41 (3): (1977), the contents of which are hereby incorporated by reference.
The reasons for the diminution in exoenzyme secretion as soon as a batch fermentation is converted into a continuous fermentation have not yet been completely elucidated. The observation that exoenzyme production in batch cultures takes place mainly in the late logarithmic and stationary phases and before sporulation, and not, in contrast, in the logarithmic phase of growth (Dancer and Mandelstam, "Criteria for Categorizing Early Biochemical Events Occurring During Sporulation of B. Subtilis," J. Bacteriol.: 411-415 (1975)), leads to the conclusion that the formation of exoenzymes, while generally constitutive, is subject to catabolite repression (Schaeffer, "Sporulation and the Production of Antibiotics, Exoenzymes, and Exotoxins," Bacteriol. Rev.: 48-71 (1969)). Catabolite repression is also suggested by the observation that the production of exoenzymes increases with the difficulty of utilization by the organisms of the carbon source in the medium (Moses and Sharp, "Intermediary Metabolite Levels in Escherichia coli," J. Gen. Microbiol. 71: 181-190)), e.g., the yield of enzyme is better when glycerol or lactose, rather than glucose is used as the carbon source.
In light of catabolite repression of this nature, there have been various proposals of multistage, continuous fermentation processes. See Tsaplina & Loginova, "Production of bacterial proteinase during continuous cultivation," in 5th INT. FERMENTATION SYMP. VERSUCHS-UND LEHRANSTALT FUR SPIRITUSFABRIKATION UND FERMENTATIONS-TECHNOLOGIE 262 (H. Dellweg ed., Berlin). But these proposed processes have proved unsatisfactory for industrial production.